Culture and Identification of Rat Bone Marrow Derived-Endothelial Progenitor Cells in Vitro

Objective To investigate methods of culturing,differentiation and identification of rat bone marrow derived-endothelial progenitor cells(EPCs).Methods The marrow mononuclear cells were gained from Wistar rat bone marrow by density gradient centrifugation,and cultured in microvascular endothelial growth medium-2.Cells were isolated with adherence screening method and differentiated to EPCs after induction.The procedure of EPCs growing and differentiating was observed.EPCs were identified by cellular morphology and cellular phaenotype and cellular function.Results After 3 days culture,cells nearly completed adhering to culture plates.After renovating culture fluid on the 4th day,cells started to form colonies.While cultured for 7 days to 8 days,cells were differentiated into endothelial-like cells and formed typical cobblestone-like structure.On the 7th day and 10th day,adherent cells were identified positive for CD34,Flk-1 and CDl33 by immunofluorescence staining.Observation using fluorescence microscopy,the double-positive staining with DIL-ac-LDL and FITC-UEA-1 cell population was above 75 percent among adherent cells.Conclusion The bone marrow mononuclear cells are gained by density gradient centrifugation,then via adherent culture in microvascular endothelial growth medium-2,EPCs are obtained and amplified in vitro.